Feline calicivirus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule

A feline calicivirus and antibody technology, applied in the field of immunoglobulin molecules and applications, feline calicivirus antibody sequences, and tetrapeptide chain molecules

Active Publication Date: 2020-10-30
青岛博隆基因工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The difficulty of solving the above problems and defects is as follows: in order to obtain a certain scale of antibody library, the first thing to do is to use RT-PCR technology to obtain a complete set of antibody genes from the body. At present, there are few reports on the gene sequence of cat origin. Verification of multiple pairs of primers for light and heavy chain variable region sequences

Method used

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  • Feline calicivirus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule
  • Feline calicivirus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule
  • Feline calicivirus antibody sequence, tetrapeptide chain molecule and immunoglobulin molecule

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of cat-derived anti-feline calicivirus single-chain antibody library

[0062] 1. Isolation of Feline Peripheral Blood Lymphocytes

[0063] According to the International Animal Immunization Manual, 5 experimental cats were immunized with vaccine. Each immunization interval was 2 weeks, and a total of 3 immunizations were performed. Two weeks after the three immunizations, the peripheral blood of the cats was collected. Take 5-10mL of fresh blood and mix evenly with whole blood and tissue diluent at a ratio of 1:1-1:2. Add cell separation solution to a 15mL centrifuge tube, and gently add an equal volume of diluted anticoagulant blood along the tube wall. The horizontal centrifuge is centrifuged at a speed of 400g-800g and a time of 15min-25min. After centrifugation, the liquid in the centrifuge tube should be divided into four layers, from top to bottom: the first layer: plasma layer; the second layer: lymphocyte layer; The third layer: separa...

Embodiment 2

[0070] Example 2 Screening of cat-derived anti-feline calicivirus single-chain antibody library

[0071] 1. Preparation of Phage Antibody

[0072] Add 1 mL of phage primary antibody library to 3 mL of XLI-Blue bacterial solution with OD600=0.5, place at 37°C for 45 min, add 6 mL of LB liquid medium containing Amp+ (100 μg / mL), and add glucose (1 mol / mL) at a ratio of 1:1000 L) Continue to cultivate, when the OD600 of the bacterial solution is 0.5, add the helper phage M13K07, place it in a 37°C incubator for 30min, and then incubate at 220rpm / min at 37°C for 1h, centrifuge the bacterial solution at 3500rpm for 10min, discard the supernatant, and use an equal volume LB liquid medium containing Amp+ (100 μg / mL) and Kana (50 μg / mL) was added with IPTG (1 mol / L) at a ratio of 1:1000, 220 rpm / min, 37°C, and shaken for 6 hours. Centrifuge the culture medium at 12000rpm for 10-20min, and collect the supernatant. Add PEG8000 at a ratio of 1:4-1:5, invert and mix well, at this time c...

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Abstract

The invention belongs to the technical field of virus antibodies, and discloses a feline calicivirus antibody sequence, a tetrapeptide chain molecule, an immunoglobulin molecule and applications thereof, and the sequences are as follows: the amino acid sequence of a heavy chain variable region SEQ ID NO: 1 and a nucleotide sequence SEQ ID NO: 3; the amino acid sequence of a light chain variable region is SEQ ID NO: 2, and the nucleotide sequence of the light chain variable region is SEQ ID NO: 4. The screening method of the virus antibody sequence comprises the following steps: preparing a bacteriophage antibody; screening the phage antibody library; identifying an anti-feline calicivirus feline phage single-chain antibody by virtue of a Page ELISA method; sending the scFv bacterial liquidwith the positive Page ELISA identification result to a sequencing company for sequencing to obtain sequences of variable regions of the heavy chain and the light chain. The invention provides support for constructing a cat-derived anti-feline calicivirus genetic engineering antibody with high affinity and low immunogenicity, and has important significance for promoting the development of cat-derived antibody drugs.

Description

technical field [0001] The invention belongs to the technical field of virus antibodies, and in particular relates to a feline calicivirus antibody sequence, a tetrapeptide chain molecule, an immunoglobulin molecule and applications thereof. Background technique [0002] At present, feline calicivirus (Felinecalicivirus, FCV) is a kind of calicivirus, which can infect many animals such as humans. The clinical manifestations are ulcers in the nostrils, mouth, skin edema, severe pneumonia, etc.; the clinical manifestations of chronic cases are damage to the nostrils and palate, low-grade fever, and secretions from the mouth and corners of the eyes. The current distribution of the virus is worldwide . [0003] At present, the clinical treatment of feline calicivirus disease mainly relies on monoclonal antibodies. Most of the commercially available antibodies are immune serum of xenogeneic animals. Although they can protect young animals and adult animals from infection, they c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/10G01N33/569C40B50/06
CPCC07K16/10C07K16/005G01N33/56983C40B50/06C07K2317/622C07K2317/56G01N2333/08
Inventor 曲雪婷尹燕博
Owner 青岛博隆基因工程有限公司
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